1. Field of the Invention
This invention relates to the art of immunological detection and more particularly, a method for the immunological detection wherein there are utilized antibodies capable of binding with analytes or substances to be detected.
2. Description of the Prior Art
Immunological detection utilizing naturally occurring or artificially prepared antibodies has merits in high selectivity and high sensitivity to analytes or objects to be detected and has thus been currently employed for the purpose of detecting analytes present in very small amounts. This detection technique has been applied, for example, to clinical examinations such as of so-called disease markers which are specifically secreted when one suffers diseases accompanied by tumors, cardiac infarction, cerebral thrombosis and the like, or also to detection of substances contained in air in very small amounts. For the detection of these markers or substances, it is usual that the sensitivity for determination of the presence or absence of bound antibody is improved by the use of radioactive isotopes or enzyme reactions. However, the use of radioactive isotope is limited to a laboratory scale test because of the danger involved therein. A number of the detection techniques utilizing the enzyme reactions, i.e. enzyme immunoassays (hereinafter sometimes referred to as EIA), have been proposed using various combinations of the reactions (R. M. Nakamura, A. Voller and D. E. Bidwell, Enzyme Immunoassays: Heterogeneous and Homogeneous Systems in Handbook of Experimental Immunology 4th Edition, Vol. 1 and Immunochemistry, Chapter 27, edited by D. M. Weir, Blackwell Scientific Publications, Oxford, 1986). These EIA methods can be broadly divided into two classes, one being directed to high molecular weight substances or proteins as analyte and the other dealing with low molecular weight analytes, e.g. haptens. Analytes to which the present invention is directed are mainly haptens. Accordingly, an enzyme linked immunosolvent assay (ELISA) which is a typical technique for haptens is illustrated with respect to experimental procedures.
The ELISA technique is generally carried out in the following procedure.
(A) A conjugate of a carrier protein such as, for example, bovine serum alubmin (BSA) analyte or its derivative introduced with a functional group is dissolved in a buffer to form an antigen solution. The antigen solution is added to a microplate (e.g. a polyvinyl chloride or polyethylene 96 well plate) in an amount of 100 .mu.L/well and incubated at 20.degree. C. overnight.
(B) Blocking
A phosphate buffered solution of BSA having a pH of 7.5 is added in an amount of 250 .mu.L/well, followed by allowing to stand at room temperature for 0.5 to 2 hours and washing with a buffer or pure water 3 to 5 times.
(C) Antibody Reaction
A solution of an analyte is added to the well plate, to which an antibody solution is further added while shaking. After keeping it at normal temperatures for 3 to 5 hours, the antibody solution is removed by means of an aspirator, followed by washing with a buffer or pure water three to five times.
(D) Second Antibody Reaction
A solution of an anti-mouse IgG antibody against an antibody labelled with an enzyme such as, for example, peroxidase is added to the washed mixture and allowed to stand at normal temperatures for 0.5 to 2 hours, followed by washing with a buffer or pure water three to five times.
(E) Reaction of Substrate and Its Termination
A color developer such as, for example, o-phenylenediamine is dissolved in a buffer to obtain a solution. Immediately before its application, the solution added with 30% hydrogen peroxide is further added as a substrate solution, followed by color developing reaction at room temperature. Five to twenty minutes after the addition, the reaction is terminated with sulfuric acid.
(F) Measurement
An absorbance at 492 nm is measured by the use of a spectrophotometer for microplate. The absorbance becomes weaker for a larger amount of the analyte, from which the analyte can be quantitatively determined.
As will be apparent from the above, the ELISA technique has the advantage in treating a number of specimens at the same time, but requires the complicated procedure, which in turn requires the skill on the part of a tester. In addition, this technique is difficult to automate, coupled with the further problem that it takes a very long examination time of not shorter than 5 hours. These problems are true of the other EIA techniques set out in the literature indicated above.
The reason why these EIA techniques take a long time and much labor is considered as follows. In most cases, these techniques make use of a procedure of separating antigen-bound antibody and free antibody from each other (B-F separation). This procedure involves either a heterogeneous reaction where solid and liquid phases coexist or a homogeneous reaction in solution where proteins including antibodies react with each other. In either case, the reaction speed is very slow and thus entails an increase of the detection time.